11 beta hydroxylation of steroids by botrytis cinerea



11 BETA HYDROXYLATION OF STEROIDS BY 7 BOTRYTIS CINEREA Louis I. Feldman, Hillcrest, and William Shardlow Allen,

Pearl River, N. Y., assignors to American Cyanamid Company, New York, N. Y., a corporation of Maine No Drawing. Application June 16, 1955,

Serial No. 516,046 6 Claims. (Cl. 195-51) This invention relates to the oxidation of steroids, especially to the introduction of an hydroxyl group at the ll-position in the ER-configuration. In particular, the invention is concerned with Kendalls Compound F (hydrocortisone, M-pregnene-l1B,l7a,2l-triol-3,20-dione). 7

Recently, a number of steroids having an hydroxyl group at the l-l-position have become important thera peutic agents in the treatment of diseases. Among these steroids is Kendalls Compound F. Processes of producing this substance from ll-desoxy steroids have been developed, but unfortunately suitable intermediates are scarce, and the processes of introducing an hydroxyl group at the ll-position are unsatisfactory for various reasons. The present invention provides an eflicient means of introducing an hydroxyl group at the ll-position of Reichsteins Substance S to produce Compound F.

Reichsteins Substance S, which is A -pregnenel7a,21-

diol-3,20-dione, is a commercially available product made from diosgenin, the original source of which is various species of Mexican yams of the family Dioscoreacea-e. The process of the present invention is believed to represent an advance over prior methods of oxidizing steroids at the ll-position because of its freedom from side reactions and high yields. It appears that the process of the present invention yields primarily the desired steroid product, which can be readily recovered by extraction processes such as will be described hereinafter.

The process of the present invention is a fermentative oxidation using organisms of the class Fungi imperfecti, which. in general reproduce asexually. The. genus Botrytis is useful in the present process. Several isolates of Botrytis species of the cinerea type [Botrytis cinerea (ATCC 7020)] have been classified also as Sclerotinia elianaMycologia 45, p. 422 (1953). Several other Botrytis species have also been classified as Sclerotinia.

In carrying out the process of the present invention,

rllce ing peptones, amino acids, and many other available proteinaceous materials which have been found suitable in fermentation processes for the supportof growth of i fungi. Various inorganic sources of nitrogen, includ1ng urea, ammonium salts, nitrates, and the like, may also be included in the medium as a source of assimilable nitrogen to provide a favorable growth substrate for the organism.

a fungusof the Botrytis genus, such as Botrytis cinerea, Botrytis peoniae, and/or other Botrytis species, is culti- I 'va-ted aerobically in a suitable nutrient medium and allowed to act upon Substance S or an ester thereof, such as acetate, or a related ll-desoxy steroid. During growth of the organism under favorable conditions, an hydroxyl group is introduced into the llposition. The exact mechanism of this oxidationis obscure, but itis the result of enzymes produced by the organismas it grows. A suitable nutrient medium contains a soluble source of carbon, nitrogen, and mineral elements.

Sources of carbon include corn starch, molasses, maltose,

dextrose, sucrose, xylose, galactose, glycerol, mannitol', and various organic acids, such as citric acid, malic acid, acetic acid, and various natural products containing carbohydrates, such as corn steep liquor, soybean meal, cotton seed meal, and many other available materials which have been usedheretofore as a source of carbon in fermentation processes. Usually, a variety of the above can be employed in the medium with good results.

*Suitable sources of nitrogen include some of the abovenamed materials, such as corn steep liquor, soybean Cally digested proteins, and degradation products, includ- Ordinarily, much of the mineral requirements of the fermentation are present in the crude materials that are used to furnish sources of carbon and nltrogen or occur in the waterthat is used in the process. Generally,

however, it is advisable to supplement the minerals normally present with added amounts to obtain a maximum growth of the fungus. Cations and anions which may' be desirable in added amounts include the phosphate,

sulphate, chloride, sodium, potassium, magnesium, iron, calcium, cobalt, manganese, and various others. Inasmuch as the use of mineral elements in supporting the" growth of fungi is fairly well understood, elaboration "appears to be unnecessary.

The ll-desoxy steroid may be added to the medium prior to, or one or two days after, inoculation. The fermentation is then continued for a period of 1-4 days or more to efiect oxidation of the steroid.

To prepare inocula, 5-10 ml. of sterile water is used to suspend the surface growth-of an agar test tube slant' The resulting suspension of spores and usually carried out at about 20-- C. 1

Duringrthe. fermentation process aeration isprovided by forcing sterile'air through the medium at a rate ap-' proximating /s 'to 2 volumes of air per volume of medium per minute.

as needed. I

The steroid to be oxidized is added to the fermentation in solution or in finely divided form. A preferred method isto .dissolve a steroid in methanol or other water-' miscible solvents and add it to the fermentation medium at the desired stage of the process. Although the steroid may precipitate-from solution when so added, it is dispersed throughout the medium as a fine suspension and becomes readily available to the organism for oxida tion.- The amount of steroid addedto the fermentation may vary considerably, but it is'generally of the order,

of 1 gram per liter of medium.

At the conclusion of the fermentation process, the

desired l'l-hydroxylated steroid is recovered from the.

fermentation medium by the following procedure, which" describes in .particulara 10 ml. fermentation. This is a: l

operative for fermentations of general procedure and is various sizes.

Acetone (-150 ml.) is added to 10 ml. offermented beer, and the mixture is allowed to stand at room temperature-for a while and then filtered. f The filtrate is con centrated under reduced pressure until no acetone remains- The aqueous residue is transferred to a separatory funnel, and about 100*mL1of.

(aqueous volume, 10-15 ml.).

water is added. The aqueous solution is then extracted The contents of 2 or 3 such.

Temperatures between about 15-28 C. are preferred, with reaction Mechanical agitation is used to keep the mycelium and other insoluble materials in suspension. Anti-foaming agents, such as silicones, glyceride oils, and the like, may be added from time to time and in amounts 4 times with 20ml. portions of; methylene chloride. The 4 extracts are pooled, and the resulting solution is washed once with 2% aqueous sodium bicarbonate saturated with sodium chloride and then twice with about 50 ml. portions of saturated sodium chloride solution. The washed, e hyle e chlor e sol o s dried over anhy rous m snesiurn sulfate and filtered. ;The filtrate is concentrated on a steam bath. at atmospheric pressure to 3-5 ml., and the concentrateis transferred to a ml. volumetric flask and brought up tovolume with methylenechloride. This solution is used for characterization of steroid content as described hereinafter.

In larger scale fermentations, the crude product or prod cts may be, recovered from the. fermentation beer by.

mple solvent extraction, using a suitable watertimmisciblefiplvent, such as chlorinatedlower. hydrocarbons, alcohols. esters, ketones, etc. Further. purification and separation of steroid products from extracts may be accomplished by. methods well understood by those skilled mtheart. Separation of steroid mixtures often requires he use of chromatography.

The. process employed to identify the steroids present inthe extracted fermentation beer described above is by paper strip chromatography. The solvent system used is water-methanol-benzene prepared by shaking of approximately 50% water-50% methanol with benzene in a separatory funnel, and then allowing the two layers to separate. A portion of the lower layer is placed in an open dish on the floor of a large glass cylinder. The upper layer is the mobile phase and is used to fill the troughshaped well within the cylinder. A standard steroid solution is prepared by dissolving 10 mg. of each of the following steroids in 10 ml. of methylene chloride:

Reichsteins Substance S Cortisone Hydrocortisone Il-eoi-hydrocrtisone (Other steroids can be included in the standard solution when appropriate) At least one standard steroid solution is chromatographed simultaneously each time an unknown solution is tested. Exactly 0.025 ml; of the standard steroid solution is applied to the paper strip at the starting line, 4 inches from the upper end of the strip, which is folded over the edge of the'trough and immersed in the mobile phase within. The strip is then developed for 2-4 hours at 37 C. Similarly, 0.1 ml. of the unknown solution is applied to another strip which is then folded into the same trough and developed with the steroid standard strip. Thetrough permits development of'many strips simultaneously. After proper development of the paper strips, they are removedfrom the apparatus and air-dried. After drying, the strips are sprayed with an alkaline solution of BlueTetrazolium, which generates color with steroids which contain a ketol side-chain. Color-developedstrips are alined with at least one standard strip and evaluated. The different steroids can be identified by their positionson the strips.

The specific examples which follow illustrate in detail the oxidation of Reichsteins Substance S and purification of the resulting hydrocortisone.

PEEL-4.0.

Twelve liters of the above medium containing three grams of Reichsteilfs u st .8, ster zed. in. a. ZQ-litsr Ex l 1 i d Percent Corn steep liquor 1.25 Mannitol 1.0 (NI-14).:HPO4 0.2 KH2PO4 0.15 'KHzHPOs 0.05 MgSQy'YHzO 0.025 NaCl 0.2 Wisconsin A-Z salts solution 0.1 Water: to volume.

bottle was inoculated with 200 ml. of a 3-day mycelial growth of Botrytis cinerea (Lederle Culture No. N-51). The fermentation was carried out at 28 C. for 240 hours, after w i h. ime paperstrip an y es. indicated a, conversion to hydrocortisone of about 20% The resultant fermentation mixture was filtered and the 'mycelium washed with 2 liters of acetone. This extract was combined withthe beer and the acetone. evaporated under reduced pressure. Thebeer'was then extracted with 4 successive 2liter, volumes of methylene chloride; the extracts were combined and washed 2 times with saturated saline. After drying 'over anhydrous sodium sulfate, the extract. was evaporated under reduced pressure, yielding 4 g. of an .oily residue.

The residue wasthen dissolved'in a. portion ofthe sol: vent phase from he system; ethyl. acetate, 4; petroleum ether (boiling point -1100,), 2; methanol, 3.; water, 2;, and partitioned on a, column consisting. of 220 g, of diatomaceous, earthand 110. g. of the waterphase from the above system. The eluted, fractions containing the de-. sired steroid were, combined and evaporated to dryness under reduced pressure. Crystallization of the residue from acetonepetroleum, ether .(60-.70) gave crystals. melting at 2.13-2.16 C.., [a] .l.162 (ethanol); ultraviolet spectrum:

31 0 s, .309) The infrared. abSQ Ption Spectrum was identical to that of aniau thentic sample of At-pregnenedl-beta l7-alpha 21-triol-.3,20?dion e (hydrocortisone) Example 2 ml. of n ediurnas described above in Example 1 in a soogmr'nnenmsy r flask was inoculated with 1 ml. of a spore suspension of Botry'tis ciuered (L'ederle Culture No. N-51) grown on TSAagar. -[TSA agar is made up of 3% trypticase soy broth, (Baltimore Biological Labo;

ratory), 5% gilycerine, 0.3% beef extract, and 1.5 agar-l After 5 8, hours shaking at 28 C., onen' l. of this inoculum was used to inoculate 100ml. shaker tubes containing ,10 ml. of sterile medium, as in Example 1, and 2 mg. of fReichsteins Substance S. One tube was incubated for. 96 hours, and after that time paper chromatographs indicated the prsence 'of about 1 mg. of hydroc0rtis'one (a''yie.1d.of50%). w

Ex mn w Percent Cerelose 1.0 Yeast extract 0.1 NaCl 0.25 ilee'f'extracL, 0.4 Pept'one 0.4

pH adjusted to pH 7 with NaOH.

Twelve liters of the above medium in a 20 liter bottle 7 equipped with astirrer and an aerator was inoculated with 300ml. of a 3-day old mycelial growth of Botrytis cinerea (Ledcrle Culture No. N+51 61). The fermentation was carried out at 21? C. Three grams of'Reichsteins Substances were added to the bottle prior to autoclaving. The fermentation was followed periodically ,by paper strip assay, and the bottle harvested at 72 hours, when point 217-218 C. The infrared spectrum was identical to that of an authentic sample.

Example 4 In a further experiment under comparable conditions as described in Example 2, using the medium of Example 3, Reichsteins Substance S acetate was substituted for Reichsteins Substance S, and gave a similar yield of hydrocortisone, as indicated by chromatographic analysis. Botrytis cinerea (Lederle Culture No. N-S 1-61) was employed as the microorganism and the fermentation was carried out at 21 C.

Example 5 In a further experiment under conditions similar to those used in Example 4, several isolates to Botrytis cinerea, Botrytis peoniae and three other Botrytis species converted Reichsteins Substance S into hydrocortisone 3. A process which comprises the step of subjecting Reichsteins Substance S to the enzymatic action of the fungus Botrytis cinerea and recovering therefrom hydrocortisone.

4-. A process which comprises the steps of inoculating a nutrient medium containing assimilable carbon, nitrogen, and mineral salts and Reichsteins Substance S with the fungus Botrytis cinerea, and permitting the fermentation to proceed until a substantial amount of the Substance S has been oxidized at the ll-position to hydrocortisone.

5. A process which comprises the steps of inoculating a nutrient medium containing assimilable carbon, nitrogen, and mineral salts and the acetate of Reichsteins Substance S With the fungus Botrytis cinerea, and permitting the fermentation to proceed until a substantial amount of the Substance S has been oxidized at the ll-position to hydrocortisone.

6. A process of oxidizing a steroid selected from the group consisting of Reichsteins Substance S and esters thereof which comprises the step of subjecting said steroid to the oxidative fermentation action of Botrytis cinerea and recovering the li-beta-hydroxylated steroid therefrom.

References Cited in the file of this patent UNITED STATES PATENTS 2,602,769 Murray July 8, 1952 

1. A PROCESS WHICH COMPRISES THE STEP OF SUBJECTING A MEMBER OF THE GROUP CONSISTING OF REICHSTEIN''S SUBSTANCE S AND ESTERS THEREOF TO THE OXIDATIVE FERMENTATIVE ACTION OF BOTRYTIS CINERA AND RECOVERING THEREFROM HYDROCORTISONE. 